Biofuels are produced using microbial fermentation processes. Fermentation is one of the oldest processes known to mankind and can be used to make, interalia, ethanol (or bio-ethanol). It is cheaper to produce ethanol from petroleum feedstock while albeit more environmentally unfriendly in many regards. However, due to the concern of diminishing availability of fossil fuel, microbial production of biofuel is of significant interest. Traditionally, ethanol has been produced in batch fermentation using yeast strains that cannot tolerate high concentration levels of ethanol. Therefore, strain improvements have been investigated to obtain alcohol-tolerant strains for the fermentation process. The viability of using fermentation for industry-wide production, especially for the production of ethanol, depends on being able to control the fermentation process and overcome some of its inherent shortcomings.
All microbial fermentation processes require a source of energy (a nutrient) to feed the organisms. Typically, the carbon source is used by the microbe for its own energy production. In other words, oxidation of the carbon source provides energy for microbial metabolism.
Fermenting microbes possess varying capabilities of breaking down different carbon-based sources energy. Fermentation processes from any material that contains sugar can be used to produce metabolic products. For example materials for producing ethanol are typically classified under three types of agricultural raw materials: sugar, starch, cellulosic. Sugars, a carbon source, such as sugar cane, sugar beets, molasses or fruits, can be converted to ethanol directly. Starches, e.g., grains, potatoes, or root crops must first be hydrolyzed to fermentable sugars by the action of enzymes from malt or molds. And, cellulosic ethanol is derived from wood, agricultural residues, waste sulfite liquor from pulp and paper mills. These cellulosic materials must also be converted to sugars, in general, by the action of mineral acids or enzymes. After the simple sugar is formed, enzymes from yeast or microbes can readily ferment the sugar (the carbon source) to ethanol. Microbes are currently being engineered to breakdown cellulose directly. Oxidation is by definition the removal of electrons, and thus, those electrons need to be transferred somewhere. These electrons are transferred to an electron acceptor which undergoes reduction, and consequently, maintains the charge neutrality within the microbe. The electron acceptor has to also be regenerated to remove further electrons or be continuously replenished. However, replenishment consumes energy and is not necessary if oxygen is present for example in the ambient atmosphere. Respiratory processes in bacteria are remarkable because of their ability to use a variety of compounds, including insoluble minerals as terminal electron acceptors.
Fermentation processes inherently generate excess electrons within microbes as a result of oxidation of a carbon source by the microbe. The conversion of any carbon source, leads to the generation of excess electrons within the microbes as a result of oxidation of the carbon source. The removal of these excess electrons is a critical molecular mechanism that basically begins within the microbe and typically terminates outside the microbial cell. Examples of some of the molecular mechanisms inherent to microbes include but are not limited to oxygen reduction (transfer to oxygen), organic metabolites reduction, and mineral reduction such as iron or manganese.
Microbes generally have developed evolutionary pathways to dispose of or “remove” excess electrons. Certain microbes even expend energy by secreting electron shuttling compounds to rid the excess electrons that are generated. Further, it has been introduced that microbes produce pili or nanowires for the purpose of removing excess electrons. Various proteins are also believed to be involved in the process of electron transfer by microbes to insoluble materials (e.g., minerals). These proteins include, but are not limited to, outer membrane proteins and periplasmic and extracellular cytochromes. These naturally occurring mechanisms, however, have a kinetically slow rate of removal of the excess electrons.
Many types of bacteria are capable of ethanol formation. However, the microbes will also produce other end products (metabolites) besides ethanol. There is a collection of bacteria that are known to produce primarily ethanol, including but not limited to Clostridium sporogenes, Clostridium indolis, Clostridium sphenoides, Clostridium sordelli, Zymomonas mobilis (“Z. Mobilis”), Zymomonas mobilis Sp. Pomaceas, Spirochaeta aurantia, Spirochaeta stenostrepta, Spirochaeta litoralis, Erwinia amylovora, Leuconostoc mesenteroides, Streptococcus lactis, Sarcina ventriculi. It has been reported that the Z. mobilis is a better candidate for industrial alcohol production, and that Z. mobilis further possesses advantages over yeast with respect to ethanol productivity and tolerance. Z. mobilis, a facultative gram-negative bacterium, is one of the organisms that can be used in large-scale bio-ethanol production.
Bacteria generally grow in nutrient deficient conditions (e.g., soil) and thus, nutrients can be a limiting factor in their metabolism. However, in industrial processes, bacteria are grown in a nutrient rich medium and the naturally occurring molecular mechanism of the bacteria for removing electrons remains a rate limiting factor that may contribute to the inefficiency of the fermentation process to produce ethanol.
One possible solution is to manage glucose limitations by discontinuous feeding of glucose solution. However, a continuous process using co-immobilized amyloglucosidase (AMG) is believed to be a better fermentation process for Z. mobilis providing operational stability for over 40 days. Other proposed solutions, include the use of mixed culture of different ethanologenic strains to improve productivity. However, in any case, the continuous or batch methods still rely upon the innate mechanism of the microbe itself and does not address the ability of the microbe to dispose of excess electrons.
Electron acceptors such as anthraquinone disulfonic acid (AQDS) when added as an external (exogenous) electron shuttling agent can also result in a faster kinetics of removing electrons when monitored by the solubilization of an iron oxide. Thus, the mechanism for removing electrons can be altered. There are a number of known electron acceptors, for example, dissimilatory iron reducing microorganisms electron acceptors such as oxygen, metals, extracellular quinines, quinones (e.g., AQDS), sulfur compounds, nitrate, fumarate, chlorinated compounds, and electrodes can be used. However, it is reported that the addition of organic redox mediators does not always enhance the reduction of insoluble iron to the same extent. Thus, it is not certain how or albeit even if an electron acceptor will have an effect on reduction of metal or as an electron acceptor in general for a given microbe in the reduction of metal. It is also reported that electric potential or current applied to cells, tissues and organisms results in the ability to stimulate their metabolic pathways (e.g., cell growth and glucose breakdown or utilization). This has been demonstrated for a high active strain of Streptomyces noursei ZIMET 43716, and yeast. Further, there are mutants of Shewanelia putrefaciens that are unable to respire on humic substances.
Research has also been carried out for optimizing fermentative organisms, low-cost substrates, and environmental conditions for fermentation to occur. As an illustration, a strain improvement program recognized the need to obtain an alcohol-tolerant strain for the fermentation process. The cells are recycled in fermentation by immobilization in a suitable matrix.
It has also been reported that microbial fuel cells (MFCs) can harvest electricity from an organic matter stored in marine sediments. Additional studies related to these systems, has introduced the concept that microorganisms conserve energy to support their growth by completely oxidizing organic compounds to carbon dioxide with direct, electron transfer to electrodes suggesting that self-sustaining MFCs are feasible. It is also reported that many applications of MFCs rely on the selection of electricity-producing microorganisms from the natural community of microbes in the organic source material. However, the exploration of these systems has failed to recognize an effect, if any, that these same electricity-producing microorganisms have in metabolic product generation based on glucose utilization.
Typically, the microbes for producing electricity in MFCs are different types of microbes than the fermenting microbes for generating metabolic products based on glucose utilization. The electricity producing microbes degrade glucose to carbon dioxide while the microbes used for metabolic product generation degrade glucose to the level of a simpler substance such as ethanol.
One of the main bottlenecks experienced with MFCs is the electron transfer from the bacteria to the anode. A transfer resistance is caused by either oxidizing a compound at the anode surface or reducing a compound at the bacterial surface or in the bacterial interior surface that can require a certain energy to active the oxidation reaction. Transfer resistance results in potential losses between the bacteria and the electrodes, generally referred to as over potentials. Thus, the build up of excess electrons is a phenomenon recognized in the field of MFCs.
There remains a need to further expand and enhance the mechanism for metabolic product generation derived from glucose utilization as well as for current generation. To this end, the present invention relates to a method of removing electrons by providing a conduit for the transfer of electrons produced during the fermentation process and thereby enhancing the rate of production of metabolites derived from glucose utilization in addition to the generation of useful electricity.